This invention is in the field of molecular biology. In particular, this invention relates to the isolation, purification, cloning and expression of plant xylanases, nucleotide sequences encoding plant xylanases and further relates to the control of fruit and vegetable softening, as well as the control of growth, abscission and dehiscence in plants.
The ability to market high quality fruits and vegetables is dependent upon on the control and regulation of several interrelated factors. These factors include the regulation and control of plant growth, abscission and dehiscence processes as well as the control of softening of fruits and vegetables.
Xylans are a significant component of plant cell walls and are regarded as hemicellulose. Xylanases are enzymes involved in the breakdown and release of xylose oligomers from cell wall xylans. Xylanases are grouped with other cell wall hydrolases that degrade hemicelluloses and are referred to as hemicellulases. However, the role of hemicellulases in wall softening has not received much study (Lashbrook et al., 1998). For convenience sake, each of the references and articles cited herein are listed with their complete citations at the end of the application before the claims section.
Abscission is a process involved in the loss of vegetative buds, branches, roots, sepals, petals, anthers, ovaries, fruit, leaves, fruit, and flowers in plants in response to developmental cues (e.g. Infertile flower, leaf senescence, ripe fruit, etc) and environmental stresses (e.g. Frost, insufficient light, high temperature, defense reaction to herbivore, fungi and bacteria) (Sexton, 1995). As such, control of abscission is critical to maximizing yields and fruit and vegetable quality. Abscission is an active metabolic process resulting in the weakening of the cell walls, of anything from 1 to 20 rows of cells in genetically determined abscission zones. Cell wall hydrolases such as polygalacturonase, glucanases, pectin methyl esterase, cellulases and hemicellulases are thought to be involved.
Dehiscence is the spontaneous and sometimes violent opening of a fruit, seed pod, or anther to release the seed or pollen. This process shares a number of feature in common with abscission. Both dehiscence and abscission are a consequence of cell wall dissolution at a predetermined position. Cell wall hydrolases such as glucanases, polygalacturonases and hemicellulases are possibly involved.
During the final stage of fruit development, ripening occurs that involves a number of changes and an important change is softening. This softening is thought to be the result of cell wall modification due to the activity of cell wall hydrolases. These hydrolases are developmentally regulated during fruit ripening. Fruit and vegetable ripening and associated cell wall changes often involves increases in exo- and endo-polygalacturonase, pectin methyl esterase, cellulase, glucanase, xyloglucanase, xyloglucan endo-transglycosylase and xylanase activities. Transgenic plants with antisense constructs to many of these enzymes to inhibit activity have not reduced fruit softening (Arrowsmith and de Silva, 1995; Brummell et al., 1994; Giovannoni et al., 1989; Tieman et al., 1992). Plants with reduced xylanase activity have not, as yet, been created.
Plant cell wall breakdown is important element in the loss of structural integrity and hence plays an important role in growth, abscission, dehiscence and fruit softening. There is an overlap between the cell wall changes that occur during fruit softening and the wall changes that occur during growth, dehiscence and abscission (Taiz, 1984; Lashbrook et al., 1994; Kalaitzis et al., 1997; Lashbrook et al., 1998; McManus et al., 1998; Peterson et al., 1996; Jenkins et al., 1996; Meakin and Roberts, 1990; Jenkins et al., 1994; Osborne, 1989; Fischer and Bennett, 1991). The processes all involve coordinated wall breakdown and in the case of dehiscence and abscission involve wall separation. While much is known about abscission, dehiscence and fruit ripening processes generally in plants, little is known about the role of plant xylanases in growth, abscission, dehiscence and fruit softening in these processes.
There is thus a need to identify plant xylanase genes and gene products and to determine their role in growth, abscission and dehiscence as well as fruit and vegetable ripening and softening. In particular, there is a need to isolate, purify and clone xylanase genes and gene products so that xylanase activity may be controlled and regulated in plants.
In order to meet these needs, the present invention is directed to purified, isolated, sequenced and cloned plant xylanase. The present invention is further directed to transgenic plants expressing cloned xylanase genes. In the transgenic plants of the invention, dehiscence and abscission processes and/or fruit and vegetable ripening processes are controlled and/or regulated.
The xylanase DNA of the invention may be isolated from any plant, preferably a dicotyledenous plant. Representative dicotyledenous plants include but are not limited to papayas, tomatoes, eggplant, potato, cabbage, broccoli, cauliflower, celery, mango, pears, plum, papayas, melons, cucumbers, avocados, peaches, apples, apricots, grapes, cherries, plums, nectarines, kiwi, raspberry, strawberries, lettuce, cranberry, sugar beet, tobacco, walnut, almond, cotton, sesame, oilseed rape, flax, soybeans, peas and the like. Representative monocotyledenous plants include, but are not limited to banana, pineapple and the like.
In general, the invention features substantially pure xylanase DNA or protein obtained from a plant, more particularly a dicotyledenous plant, most particularly a papaya. The xylanase protein of the invention is capable of hydrolyzing xylan 1,4-beta-xylose-xylose bonds.
The xylanase protein of the invention comprises polypeptide sequences substantially identical to the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:25.
In another related aspect, the invention features substantially pure DNA having a sequence substantially identical to the nucleotide sequence shown in SEQ ID NO: 24 or functional equivalents thereof. Such nucleotide sequences include those shown in SEQ ID NO:26-SEQ ID NO:33. In preferred embodiments, such DNA is cDNA or is genomic DNA. As will be apparent to those skilled in the art, functional equivalents of the invention include, for example, those nucleotide sequences which encode the same polypeptide (but which by virtue of the degeneracy of the genetic code posses a different nucleotide sequence); sequence which code substantially the same polypeptide but wherein there may be more conserved amino acid substitution (i.e. the substitution of an amino acid for one similar properties); sequences which encode substantially the same polypeptide (which preferably share 50% amino acid identity and more preferably 60% identity) but wherein there may be one or more amino acids deletions or truncations; and sequence which hybridize under standard conditions to the complete nucleotide shown in SEQ ID NO: 24. A functional equivalent would be an antisense nucleotide sequence. In a related aspect, the DNA of the invention may be linked to a heterologous nucleic acid. Such heterologous nucleic acide can be any nucleotide sequence different from that of the xylanase DNA of the invention. The heterologous DNA may be a promoter. In related aspects, the invention also features a vector and a cell (e.g., a plant) which includes such substantially pure xylanase DNA. In various preferred embodiments, the vector-containing cell is a prokaryotic cell, for example, E. coli or Agrobacterium or, more preferably, a plant cell.
The invention is further directed to isolated and purified oligonucleotides having nucleotide sequences substantially identical to those sequences selected from SEQ ID NO:5-SEQ ID NO:23.
The invention also provides a method for isolation from a plant a DNA sequence encoding an xylanase comprising, probing a DNA library prepared from plant tissue with oligonucleotide probes comprising a conserved sequence from plant xylanase, particularly papaya endo-xcex2-1,4-xylanase cDNA. The library can either be genomic or cDNA. The most preferred nucleotide sequences for papaya are xyl-31, xyl-32, xyl-41, xyl-42, xyl-43 and xyl-44, SEQ ID NO:18-SEQ ID NO:23. The preferred sequences for general isolation from other plant species for those skilled in the art would be xyl-11 to xyl-23, SEQ ID NO:5 to SEQ ID NO:17.
The present invention further includes multiple types of xylanase nucleic acid constructs including (1) xe2x80x9csensexe2x80x9d constructs encoding xylanase proteins, which can increase or reduce the expression of xylanase in plant species and (2) xe2x80x9cantisensexe2x80x9d constructs containing DNA, which can be used to produce xylanase antisense RNA to reduce expression of xylanase in plants. Optimal amounts of antisense RNA in transgenic plants will selectively inhibit the expression of genes in these plants which are involved in xylanase activity. The constructs may include nucleotides capable of encoding peptides having sequences such as SEQ ID NO:4 and 25.
Some of these constructs will direct constitutive production of transcripts. Other constructs will direct expression in specific organs and/or specific tissue layers of the transgenic plant. These organs will include leaves, petioles, stems, flower organs, seeds, fruits or photosynthetically active parts of the plant. Tissue layers will include but may not be restricted to the epidermis and adjacent cell layers. Such organ and tissue specific expression can be directed by organ and tissue specific promoters.
The present invention also provides recombinant cells and plants containing these constructs. The constructs are introduced into recombinant plants by transformation.
In one embodiment, the first category of DNA constructs include: a promoter selected from but not limited to constitutive, tissue-specific, fruit-specific, cell-type specific, seed-specific, flower-specific, fruit-specific, epidermis-specific promoters, a promoter specific to cell layers adjacent to the epidermis or a promoter specific to photosynthetically active plant tissues, which functions in plant cells to cause the production of an RNA sequence. In this embodiment, the DNA coding region sequences encode proteins which can be used to increase the activity of plant xylanase in transgenic plants. The DNA coding region will further include a region 3xe2x80x2 to the coding regions the 3xe2x80x2 nonranslated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3xe2x80x2 end of the RNA sequence promoter.
In another embodiment, a second category of DNA construct will include a constitutive promoter, seed-specific, flower-specific, fruit-specific, abscission zone-specific, dehiscence-zone specific, epidermis-specific promoter, a promoter specific to cell layers adjacent to the epidermis or a promoter specific to photosynthetically active plant tissues, which functions in plant cells to cause the production of an RNA sequence. The DNA construct will also include DNA sequences which can produce antisense RNA molecules. These RNA molecules can selectively inhibit the accumulation of transcripts encoding proteins which encode plant xylanase thereby reducing xylanase expression and activity.
In accordance with another aspect of the present invention, there is provided a method of producing genetically transformed plants which express a gene or genes involved in xylanase activity. In this method, a recombinant, double-stranded DNA molecule is introduced into the genome of a plant cell. In this embodiment, the DNA sequence will include a promoter which functions in plant cells to cause the production of an RNA sequence in flowers, seeds, fruit, other plant tissues or organs achieved, for example, by use of tissue specific promoters to regulate transcription of the introduced sequence. In addition, the sequence will include a DNA coding sequence encoding proteins involved in xylanase activity in plants. Alternatively, the sequence will be a template to the synthesis of antisense RNA inhibiting xylanase activity. The DNA sequence will also include a 3xe2x80x2 non-translated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3xe2x80x2 end of the RNA sequences. The method also includes obtaining transformed plant cells and regenerating from the transformed plant cells genetically transformed plants. The DNA sequence can be introduced into a plant by a sexual cross.
The present invention is also directed to transgenic cells such as bacterial, yeast, fungi, plant and mammalian cells expressing the DNA sequences of this invention. The present invention is also directed to purified xylanase protein isolated from the transgenic cells of the invention. The present invention is also directed to purified antibodies directed to the xylanase protein of the invention. The antibodies may be monoclonal or polyclonal.